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C36S/C118V/A152C/C160S/C197S/C257S/C293S/C204V
introduction of a Cys residue in a mutant lacking all endogenous Cys residues. Mutant achieves maximum specific disulfide cross-linking efficiency. The resulting construct is active and, when mixed with a chemically modified RNA, yields crystals of an enzyme-RNA complex
C36S/C118V/C160S/C197S/C257S/C293S/C204V
mutation of all seven endogenous cysteine residues. Mutant is able to bind RNA at a level similar to that of wild-type, but no longer forms nonspecific disulfide cross-links with the modified RNA
D167N
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strong reduction of catalytic efficiency
D167N/N202A
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sharp decrease in catalytic efficiency
D167N/T317G
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sharp decrease in catalytic efficiency
D167N/V247R
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sharp decrease in catalytic efficiency
F100D
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strong reduction of catalytic efficiency
G203H
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strong reduction of catalytic efficiency
K228A
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increase in Km for ATP
K232A
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increase in Km for ATP
N202A
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high Km value for RNA, strong reduction of catalytic efficiency, incorporates etheno-ATP and N1-methyl-ATP better than wild-type, but incorporates N6-methyl-ATP poorly
N239A
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kcat for ATP is reduced fourfold
R199A
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reduces kcat for RNA about 30-fold
T317G
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increase in Km for ATP, tolerates etheno-ATP and GTP, rejects N6-methyl-ATP
V156A
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strong reduction of catalytic efficiency
V247R
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kcat for ATP is reduced 2000-fold, strong reduction of catalytic efficiency
Y237A
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increase in Km for ATP
D170A
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D212A
mutant enzyme forms specifically incorporated A-residues with a fidelity comparable to that of the wild type enzyme
D214A
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D79A
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disparity between Diphosphate release and AMP incorporation
D88A
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D90A
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
E211A
mutant enzyme forms specifically incorporated A-residues with a fidelity comparable to that of the wild type enzyme
G74A
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disparity between Diphosphate release and AMP incorporation
G74P
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disparity between Diphosphate release and AMP incorporation
R215A
the mutation leads to a dramatic loss in nucleotide specificity and the formation of poly(N) tails
D237N
loss of activity. A heterodimer in which one monomer is active and the other is not retains activity
K112E
disruption of a a continuous positively charged surface formed upon dimerization, decrease in activity
K76E/K80E/K81E
disruption of a a continuous positively charged surface formed upon dimerization, decrease in activity
R272E
complete loss of activity
D124A
the mutant shows strongly reduced activity compared to the wild type enzyme
D126A
the mutant shows strongly reduced activity compared to the wild type enzyme
D325A
mutation of one of the conserved Asp residues in the active site, complete loss of activity. The mutant protein gives better quality crystals than the wild-type enzyme
D90A/D92A
catalytically inactive
E200A
the mutant shows strongly reduced activity compared to the wild type enzyme
G107A
the mutant shows strongly reduced activity compared to the wild type enzyme
H259A/K260A/I261A
mutation in beta-arm, mutant remains dimeric
H259A/K260A/I261A/H294A/F295A/P297A
mutations simultaneously disrupt both areas of contact in the dimer interface, mutant is a stable monomer in solution, complete loss of activity
H294A/F295A/P297A
mutation in helix alphaE, mutant exists in a monomer-dimer equilibrium
K560E
mutation in the C-terminal basic motif. About 20% of wild-type activity
S108A
the mutant shows strongly reduced activity compared to the wild type enzyme
Y221A/F222A
mutation in helix alphaB, mutant exists in a monomer-dimer equilibrium
D114A
polyadenylation-defective mutant
D90A/D92A
catalytically inactive
S537A
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the mutant catalytic domain is not phosphorylated by ERK
V498Y/C485R
the mutant is unable to bind Fip1 but retains full polymerase activity
Y224S
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12-fold increase in the Km for oligo(A)14 in comparison with wild-type enzyme, 2.8-3.6-fold decrease in Vmax
D170P
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D170P
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no detectable AMP incorporation
D214P
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D214P
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no detectable AMP incorporation
D88P
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D88P
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no detectable AMP incorporation
D90P
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diphosphate release equivalent to AMP production, 5-30% of wild type activity
D90P
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no detectable AMP incorporation
D154A
crystallization data, closed conformation with extensive interactions between substrates and all three polymerase domains
D154A
the mutant has a nearly identical melting temperature as wild type PAP
K215A
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74- and 56-fold increase in the Km for oligo(A)14 in comparison with wild-type enzyme, 2.8-3.6-fold decrease in Vmax
K215A
2- to 4fold increase in Km values
K215A
the mutant has a nearly identical melting temperature as wild type PAP
N189A
residue bridges the N and middle domains in the closed state
N189A
the mutant has a nearly identical melting temperature as wild type PAP
N226A
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74- and 56-fold increase in the Km for oligo(A)14 in comparison with wild-type enzyme
N226A
mutation affects the equilibrium between the open- and closed-domain forms of the enzyme
N226A
the mutant has a nearly identical melting temperature as wild type PAP
Y224F
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30-fold increase in the Km for oligo(A)14 in comparison with wild-type enzyme, 2.8-3.6-fold decrease in Vmax
Y224F
mutation affects the equilibrium between the open- and closed-domain forms of the enzyme
Y224F
altered melting temperature (44°C) compared to the wild type enzyme
additional information
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chimeras of CCA-adding enzyme and PAP were constructed
additional information
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enzyme knockout mutant, the mRNA levels of bolA, which is induced in response to many forms of stress, are reduced 2.5fold in stationary phase. Absence of enzyme enhances the RssB-mediated deltaS proteolysis specifically in starved cells
additional information
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enzyme PAP I variant with C-terminal His-tag can be phosphorylated both in vivo and in vitro. In vivo phosphorylation impairs activity of the enzyme
additional information
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reengineering of enzyme in order to function as (UG) adding enzyme. Double- and triple-mutants in residues 211, 212, 215 add 570 G residues to oligoA15 substrate
additional information
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hmtPAP knockdown mutant cells show decreased steady state levels of mtDNA-encoded proteins as well as deficient mitochondrial activities such as oxygen consumption rate
additional information
deletion variant of the C-terminal part lacking amino acids 369-551. Whereas the C terminus binds to RNA, the deletion variant shows no shift of the RNA in EMSA experiments
additional information
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deletion variant of the C-terminal part lacking amino acids 369-551. Whereas the C terminus binds to RNA, the deletion variant shows no shift of the RNA in EMSA experiments
additional information
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GLD-2 disruption does not affect the poly(A) tail elongation in oocytes
additional information
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the un-17 mutant carries a temperature-sensitive mutation in the gene encoding PAP
additional information
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deletion of C-terminal 31 amino acids has no effect, deletion of C-terminal 67 amino acids affects RNA binding, deletion of N-terminal 18 amino acids eliminates specific activity
additional information
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mutation of bovine residue N202 (equivalent to yeast N189) to alanine has essentially no effect on the apparent Km for ATP, but has a pronounced effect on the apparent Vmax, suggesting that this residue is particularly important in the recognition of the adenine of ATP
additional information
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strain lacking the Rrp6p component of the nuclear exosome accumulate polyadenylated forms of different ribosomal RNA precursors. This polyadenylation is reduced in strains lacking polynucleotide adenylyltransferase Trf5p and enhanced in strains overexpressing Trf5p